A recombinant ‘ACE2 Triple Decoy’ that traps and neutralizes SARS-CoV-2 shows enhanced affinity for highly transmissible SARS-CoV-2 variants

Abstract

The highly-transmissible SARS-CoV-2 variants now replacing the first wave strain pose an increased threat to human health by their ability, in some instances, to escape existing humoral protection conferred by previous infection, neutralizing antibodies, and possibly vaccination. Thus, other therapeutic options are necessary. One such therapeutic option that leverages SARS-CoV-2 initiation of infection by binding of its spike receptor binding domain (S RBD) to surface-expressed host cell angiotensin-converting enzyme 2 (ACE2) is an ACE2 decoy that would trap the virus by competitive binding and thus inhibit propagation of infection. Here, we used Molecular Dynamic (MD) simulations to predict ACE2 mutations that might increase its affinity for S RBD and screened these candidates for binding affinity in vitro. A double mutant ACE2(T27Y/H34A)-IgG1FC fusion protein was found to have very high affinity for S RBD and to show greater neutralization of SARS-CoV-2 in a live virus assay as compared to wild type ACE2. We further modified the double mutant ACE2 decoy by addition of an H374N mutation to inhibit ACE2 enzymatic activity while maintaining high S RBD affinity. We then confirmed the potential efficacy of our ACE2(T27Y/H34A/H374N)-IgG1FC Triple Decoy against S RBD expressing variant-associated E484K, K417N, N501Y, and L452R mutations and found that our ACE2 Triple Decoy not only maintains its high affinity for S RBD expressing these mutations, but shows enhanced affinity for S RBD expressing the N501Y or L452R mutations and the highest affinity for S RBD expressing both the E484K and N501Y mutations. The ACE2 Triple Decoy also demonstrates the ability to compete with wild type ACE2 in the cPass surrogate virus neutralization in the presence of S RBD with these mutations. Additional MD simulation of ACE2 WT and decoy interactions with S RBD WT or B.1.351 variant sequence S RBD provides insight into the enhanced affinity of the ACE2 decoy for S RBD and reveals its potential as a tool to predict affinity and inform therapeutic design. The ACE2 Triple Decoy is now undergoing continued assessment, including expression by a human adenovirus serotype 5 (hAd5) construct to facilitate delivery in vivo.

Structural analysis of receptor binding domain mutations in SARS-CoV-2 variants of concern that modulate ACE2 and antibody binding

“…Our described effects on ACE2 binding and antibody evasion imparted by VoC/VoI RBD mutations are in agreement with recent reports (Chen et al, 2021;Collier et al, 2021;Dejnirattisai et al, 2021;Laffeber et al, 2021;Liu et al, 2021;Wang et al, 2021aWang et al, , 2021bWibmer et al, 2021;Yuan et al, 2021), with the exception of the enhanced ACE2 affinity conferred by E484K. Several studies have reported conflicting data surrounding the effect of E484K on ACE2 binding, where both decreased (Upadhyay et al, 2021;Wang et al, 2021c;Yuan et al, 2021) and increased (Laffeber et al, 2021;Tanaka et al, 2021) affinities are observed. A variety of biophysical techniques, spike protein domains, and ACE2 constructs were used across these studies, which could account for the contrasting results.…”

Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies

“…We also measured the efficiency of exogenously added soluble ACE2-mFc proteins to neutralize unmutated and N501Y pseudoviruses via preincubation prior to cell infection (S5B Fig) . The comparison of neutralization profiles shows that the IC 50 for neutralization of the N501Y mutant is lower, suggesting that full-length spikes bearing the N501Y mutation bind ACE2-mFc to a higher extent. Taken together with recent reports [22][23][24][25][26][27][28], these 3 results are consistent with the hypothesis that the greater infectivity of the N501Y mutant stems from improved binding to ACE2.…”

“…Our studies with the N501Y mutant are consistent with the expectation that the rapid spread of the UK variant of SARS-CoV-2 is likely due to the virus being more infectious. While there can be multiple origins for the increased infectivity, our biochemical studies suggest that the N501Y mutation results in increased ACE2 binding efficiency, a finding that has been reproduced by several recent studies [22][23][24][25][26][27][28]. Our structural studies establish the molecular basis underpinning the observed increase in ACE2 binding efficiency conferred by the N501Y mutation.…”

“…This revealed that the N501Y mutation confers a modest increase in affinity for ACE2, mainly driven by a reduction in the dissociation rate constant (k off ) (S5C and S5D Fig; S1 Table ). Notably, several studies have demonstrated that the N501Y mutation confers much larger increases (3-to 16-fold) in ACE2 binding affinity when using minimal RBD constructs [23][24][25][26][27][28]. We also measured the efficiency of exogenously added soluble ACE2-mFc proteins to neutralize unmutated and N501Y pseudoviruses via preincubation prior to cell infection (S5B Fig) . The comparison of neutralization profiles shows that the IC 50 for neutralization of the N501Y mutant is lower, suggesting that full-length spikes bearing the N501Y mutation bind ACE2-mFc to a higher extent.…”

SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

“…Tanaka et al performed a live virus neutralization assay in which they also reach 100% neutralization by using low concentration of ACE2-Ig, as opposed to our results. The reason behind the discrepancy might be in the method itself as they do not specify how the neutralization percentages were calculated and how the assay was performed [45]. Furthermore, we show that treatment with RBD-Ig using SARS-CoV-2 K18-hACE2 infected mice led to decrease in disease severity as assessed by reduced body weight, lower virus titers in the lungs, and higher survival percentage.…”